Design and characterization of decoy oligonucleotides containing locked nucleic acids

摘要

Transfection of cis-element double-stranded oligo-nucleotides, referred to as decoy ODNs, has been reported to be a powerful tool that provides a new class of antigene strategies for gene therapy. However, one of the major limitations of the decoy approach is the rapid degradation of phosphodiester oligonucleotides by intracellular nucleases. To date,m several DNA analogs have been employed to over-come this issue, but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this paper we have investigated the use of conformationally restricted nucleotides in the design of decoy molecules for nuclear transcription factor kB (NF-kB). Starting from a synthetic double-stranded oligo-nucleotide, containing the kB consensus binding sequence, we designed a panel of decoy molecules modified to various extents and at various positions with locked nucleic acids (LNAs). Our results indicate that the addition of terminal LNA bases, outside the kB sequence, to generate LNA-DNA-LNA co-polymers was sufficient to confer appreciable protection towards nuclease digestion, without interfering with transcription factor binding. Conversely, insertion of LNA substitutions in the context of the kB-binding site resulted in further increased stability, but caused a loss of affinity of NF-kB for the target sequence. However, our results also indicate that this latter effect was apparently dependent not only on the extent but also on strand positioning of the internal LNA substitutions. This observation is of great importance since it provides evidence for the possibility of tuning DNA-LNA duplexes with internal LNAs into decoy agents with improved features in terms of biological stability and inhibitory effect.